Establishment of a stockpile of primordial follicles, capable of initiating growth and potentially developing to the preovulatory stage, is a prerequisite for female reproductive success. However for non-rodent species, nothing is known about the regulation of these critical steps in gonadal development. In cattle, sheep, and other large mammals, including humans, primordial follicles form over a number of weeks during fetal life, in contrast to their synchronous post-natal development in mice and rats. In addition, in ruminant and primate species no primordial follicles leave the resting pool (activate) for several weeks after follicle formation begins, in contrast to the rapid activation of a subset of newly formed primordial follicles in neonatal rat and mouse ovaries. The ovaries of fetal cattle and sheep actively synthesize steroid hormones, especially in the period before and, to a lesser extent, during follicle formation and estradiol receptors have been localized to both somatic and gametogenic cells. We have developed experimental models in which formation of bovine primordial follicles and development of their capacity to activate (i.e., to initiate growth in response to a stimulator) occur in vitro. Our preliminary data suggest the overall hypothesis of this R21 application: that steroids produced by the fetal bovine ovary, particularly estradiol and progesterone, regulate both follicle formation and the competence of follicles to be activated. We propose three specific aims to test this novel hypothesis. In Specific Aim #1, experiments to test the hypothesis that a decline in ovarian production of estradiol, and perhaps progesterone, initiates or allows follicle formation in vivo are proposed. We propose experiments in Specific Aim #2 to test the hypotheses that ovarian steroids (estradiol and progesterone) prevent the premature activation of newly formed primordial follicles and that they do this by inhibiting or regulating the progression of prophase I of meiosis (bovine primordial follicles form before the oocyte has reached meiotic arrest in the diplotene stage of prophase I). According to our working model, the gradual decline in ovarian production of estradiol and progesterone, just before and during the time when primordial follicles are forming, first allows follicle formation to occur and then promotes the development of follicular competence to activate. In Specific Aim #3, we will begin to determine signaling pathways that subserve the effects of steroids on follicular formation and competence to activate. Together these experiments will provide new information about the regulation of a critical period in ovarian development, in a species that provides an excellent, non-primate model for human ovarian development. PUBLIC HEALTH RELEVANCE: In mammalian females, a pool of primordial ovarian follicles is formed during fetal or neonatal life and since this stockpile of follicles is used through the female's reproductive life as a source of oocytes for ovulation, it is critical for reproductive success and also a potential target for fertility enhancement or contraception. In large mammals, such as cattle, sheep and primates (including humans) the primordial follicles form when the female is a fetus and virtually nothing is known about what regulates this process. We propose to use cattle as an animal model to test the hypothesis that steroids made by the fetal ovary play a role in regulating these earliest stages of ovarian follicular development.